Generation of Monoclonal Antibodies and Characterization of Novel Antibodies by Western Blotting and Immunocytochemistry

Abstract

The development by Kohler and Milstein (1975) of murine monoclonal antibodies by somatic cell fusion techniques (i.e., the production of hybridomas) has resulted in the routine production and availability of these antibodies for research and medical purposes. The two most commonly used cells in this hybridization technique are antibody-secreting B cells from the immunized animal (which provide functional immunoglobulin genes) and mouse myeloma cells which provide the correct genes for continued cell division in culture. Any material that can elicit a humoral response, e.g., viruses, whole cells, cellular components, purified proteins, synthetic peptides, carbohydrates, can be used for the production of monoclonal antibodies. Using carefully defined conditions for efficient fusions and selection of antibody-producing hybridomas from unfused cells, monoclonal antibodies specific for a wide range of antigens have been produced. Monclonal antibodies have many advantages over polyclonal antibodies, e.g., their specificity of binding, their homogeneity (antibody produced by all the decendants of one hybridoma cell is identical) and their ability to be produced in unlimited quantities (as cell culture supernatant or ascitic fluid). A major disadvantage is that the production of monclonal antibodies is more time consuming and usually more costly than polyclonal antibody production and often a panel of monoclonal antibodies to different epitopes on the same antigen is required to give similar binding affinity as a polyclonal antibody.